LOCUS       sequence-182536-        4996 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of HA tagged ubiquitin carrying zero lysines 
            (K0).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4996)
  AUTHORS   Lim KL, Chew KC, Tan JM, Wang C, Chung KK, Zhang Y, Tanaka Y, Smith 
            W, Engelender S, Ross CA, Dawson VL, Dawson TM
  TITLE     Parkin mediates nonclassical, proteasomal-independent ubiquitination
            of synphilin-1: implications for Lewy body formation.
  JOURNAL   J Neurosci. 2005 Feb 23. 25(8):2002-9.
  PUBMED    15728840
REFERENCE   2  (bases 1 to 4996)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Neurosci.
            2005 Feb 23. 25(8):2002-9."
COMMENT     Sequence Label: pRK5-HA-Ubiquitin-KO
FEATURES             Location/Qualifiers
     source          1..4996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     74..91
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    208..229
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(244..249,250..267,268..274)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    282..298
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     287..309
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     306..322
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     306..322
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        351..730
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        731..934
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     885..905
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     931..955
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        1165..1183
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1165..1182
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     1196..1215
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1300..1309
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1312..1338
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1369..1596
                     /codon_start=1
                     /product="human ubiquitin variant in which all of the 
                     lysines have been mutated to arginine"
                     /label=ubiquitin-K0
                     /note="also known as ubiquitin-KO"
                     /translation="MQIFVRTLTGRTITLEVEPSDTIENVRARIQDREGIPPDQQRLIF
                     AGRQLEDGRTLSDYNIQRESTLHLVLRLRGG"
     regulatory      1633..1642
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1646..1780
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1683..1702)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1737..1756
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1849..2206
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1849..2040
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1872..1892)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2057..2192
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2226..2243)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2226..2242)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2235..2257)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2455..2910
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2542..2561)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2752..2773
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2927..2946)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3046..3068
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3106..3124)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3192..3296
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3297..3365,3366..4157)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3515..3534)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4328..4916
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4817..4836
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
       61 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
      121 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
      181 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
      241 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
      301 tcacacagga aacagctatg acatgattac gaattaattc gagctcgccc gacattgatt
      361 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
      421 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      481 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      541 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      601 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      661 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      721 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      781 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      841 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      901 gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg
      961 gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccg
     1021 cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg taagtaccgc
     1081 ctatagagtc tataggccca cccccttggc ttcgttagaa cgcggctaca attaatacat
     1141 aaccttatgt atcatacaca tacgatttag gtgacactat agaataacat ccactttgcc
     1201 tttctctcca caggtgtcca ctcccaggtc caactgcacc tcggttctat cgattgaatt
     1261 cccgaaccga cagtcggtct cttcaccaag gccattcgcg ccaccatggg ctacccctat
     1321 gatgtgcctg actacgcaga tctcaatggt ggtggtggtg ggtcgaccat gcagatcttc
     1381 gtcagaacgt taaccggtag aaccataact ctagaagttg aaccatccga taccatcgaa
     1441 aacgttagag ctagaattca agacagagaa ggcattccac ctgatcaaca aagattgatc
     1501 tttgccggta gacagctcga ggacggtaga acgctgtctg attacaacat tcagagagag
     1561 tcgaccttac atcttgtctt aagactaaga ggtggttgag cggccgctaa gtaagtaagg
     1621 atccccagct tggccgccat ggcccaactt gtttattgca gcttataatg gttacaaata
     1681 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     1741 tttgtccaaa ctcatcaatg tatcttatca tgtctggatc gggaattaat tcggcgcagc
     1801 accatggcct gaaataacct ctgaaagagg aacttggtta ggtaccttct gaggcggaaa
     1861 gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     1921 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     1981 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     2041 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     2101 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     2161 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tgttaacagc
     2221 ttggcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     2281 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     2341 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt
     2401 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     2461 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2521 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2581 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     2641 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
     2701 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     2761 tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttaat ttataaggga
     2821 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     2881 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
     2941 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     3001 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     3061 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     3121 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     3181 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     3241 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     3301 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     3361 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     3421 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     3481 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     3541 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     3601 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     3661 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     3721 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     3781 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     3841 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     3901 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     3961 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     4021 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     4081 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     4141 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     4201 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     4261 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     4321 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     4381 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     4441 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     4501 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4561 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4621 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4681 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4741 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4801 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4861 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     4921 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     4981 ttcctgcgtt atcccc
//
